THE EFFECTS OF URSOLIC ACID ON
NEONATAL PROGRAMMING AND ITS
POTENTIAL TO PREVENT THE
DEVELOPMENT OF METABOLIC
DYSFUNCTION IN SPRAGUE DAWLEY
RATS
Nyasha Charity Mukonowenzou
A dissertation submitted to the Faculty of Health Sciences, University of Witwatersrand, School of Physiology in fulfilment of the requirements for the degree of Master of Science
in Medicine.
ii DECLARATION
I, Nyasha Charity Mukonowenzou, declare that the work contained in this dissertation is my own, except where others have helped as indicated in the acknowledgements and the reference list. This dissertation is being submitted for the degree of Master of Science in Medicine in the Faculty of Health Sciences at the University of the Witwatersrand, Johannesburg, South Africa. It has not been submitted before for any degree or
examination at any University. I certify that all the experimental procedures used in this dissertation were approved by the Animal Ethics Screening Committee of the University of the Witwatersrand (AESC number: 2014/49/D).
……… Nyasha Charity Mukonowenzou
iii DEDICATION
To Kumbulani Yose, who saw a diamond in the rough, believed in it and polished it until it sparkled
iv CONFERENCE PRESENTATION
Data from this study was presented as a poster at the 43rd Congress of the Physiology Society of Southern Africa (PSSA), held at Khaya Ibhubesi, Free State from 6-9 September 2015. The poster was awarded the first prize in the Johnny Van der Walt student poster competition and is offered in support of this dissertation:
1. Mukonowenzou N.C., Dangarembizi R., Chivandi E and Erlwanger K.H. The effects of ursolic acid administered to neonatal rats on the subsequent development of high fructose diet-induced metabolic dysfunction (poster PJ6 –conference book of abstracts).
v ACKNOWLEDGEMENTS
“Coming together is a beginning. Keeping together is progress. Working together is success” (Henry Ford) - this research is a result of a lot of teamwork. I would like to thank the following individuals and groups for their assistance;
“A teacher affects eternity; he can never tell where his influence stops” (Henry Adams) – for the supervision and mentorship, thank you Associate Professor Kennedy Erlwanger, your influence will certainly affect eternity.
“Unity is strength…when there is teamwork and collaboration, wonderful things can be achieved” (Mattie Stepanek) – for the supervision, demonstrating the power of teamwork and opportunities I never thought possible, thank you Dr. Eliton Chivandi.
“This work is not for yourselves-kill that spirit of self, and do not live above your people, but live with them. If you can rise, bring someone with you” (Charlotte Maxeke) - as you rose, you brought me with you, for your selflessness and supervision, thank you Miss Rachael Dangarembizi.
“Alone we can do so little, together we can do so much” (Helen Keller) - for animal handling and technical assistance, I am grateful to the Central Animal Services (CAS) Staff.
“None of the things I have done would have been accomplished without a true team effort” (Nick Lampson) – you sacrificed your time, gave valuable input during presentations and shared ideas during journal club meetings. A very special thank you to my colleagues in the gastrointestinal tract and nutrition laboratory: Ibrahim Ghandi, Davison Moyo, Trevor Nyakudya, Ingrid Malebana, Nomagugu Ndlovu, Busisani Lembede, Janine De Bruin, Karabo Rathebe, Jeanette Joubert and Ninette Lotter.
“Tell me and I forget, teach me and I may remember, involve me and I learn” (Benjamin Franklin) – you told me, taught me and most importantly involved me instilling a love for learning in me. For assistance with histology assays and reagent preparations, thank you Monica Gomes.
vi
“Cultivate the habit of being grateful for every good thing that comes to you and to give thanks continuously. And because all things contributed to your advancement, you should include all things in your gratitude” (Ralph Waldo Emerson) - I am grateful for the financial assistance awarded to me by the National University of Science and
Technology (Zimbabwe), University of the Witwatersrand: Faculty of Health Science Research Board & Financial Aid Office and National Research Foundation.
“Each person holds so much power within themselves that needs to be let out. Sometimes they just need a little nudge, a little direction, a little support, a little coaching and the greatest things can happen” (Pete Carroll) – to my friends and family, for cheering me on and for all the support, I am grateful.
“To our God and Father be glory for ever and ever. Amen” (Philippians 4:20, Bible) – I would like to thank God for seeing me through it all.
vii ABSTRACT
Fructose-rich diets and the early nutritional environment have been implicated in the increase in metabolic disorders worldwide. The “two hit” hypothesis has also come under the spotlight as consequences of early nutritional interventions have been shown to appear either spontaneously or after induction by a second intervention leading to worsened disease states. Current research is exploring the potential use of pharmacologically diverse phytochemicals such as ursolic acid (UA) to promote metabolic programming thereby imparting positive health benefits later in life. This study examined the effects of early administration of UA on the subsequent development of complications associated with diet-induced metabolic dysfunction in Sprague Dawley rats.
One hundred and seven suckling, six-day old male and female Sprague Dawley rats randomly received 10 ml/kg of either 0.5% dimethylsulphoxide (control), UA, 50% fructose solution or a mixture of 50% fructose and UA orogastrically for 14 days. They were then weaned onto normal rat chow and plain drinking water on day 21. At adulthood (day 70), half the number of rats in each treatment group either continued on plain drinking water or they received a 20% fructose solution as drinking fluid for eight weeks. Food and fluid intake, body mass gain, fasting blood triglyceride, and oral glucose tolerance were assessed before termination. On termination blood and tissue samples were collected to assess the effect of UA on growth, organ morphometry, adiposity, hepatic lipid storage and surrogate markers of health.
The effects of fructose were found to be dependent on the time of intervention and sex. In males, fructose consumption in adulthood resulted in a 7% increase in body mass and a 35% increase in circulating blood triglycerides which were not observed in females. A single fructose hit and fructose consumption both neonatally and in adulthood caused increased hepatic lipid storage in females by 32% and 67% respectively. In both sexes, fructose intake in adulthood caused decreases in food intake whilst increases in fluid intake were observed in female rats (P< 0.05). Fructose consumption had no effect on glucose tolerance, visceral adiposity, organ morphometry and surrogate health markers. Neonatal administration of UA caused a 6% increase in body mass in female rats and prevented excessive fructose-induced hepatic lipid storage in both male and female rats.
viii
Although fructose administration had adverse effects in the liver, especially in female rats, neonatal intervention with UA was found to alter metabolism so as to protect against hepatic lipid accumulation. Therefore, UA is a phytochemical that shows great potential in the control of hepatic lipid metabolism and its metabolic complications.
ix TABLE OF CONTENTS COVER PAGE ... i DECLARATION ... ii DEDICATION ... iii CONFERENCE PRESENTATION... iv ACKNOWLEDGEMENTS ... v ABSTRACT ... vii TABLE OF CONTENTS ... ix
LIST OF FIGURES ... xii
LIST OF TABLES ... xiii
LIST OF ABBREVIATIONS ... xiv
CHAPTER 1 : INTRODUCTION ... 1
1.1 Dissertation structure and study background ... 2
1.1.1 Dissertation structure ... 2
1.1.2 Background ... 2
1.2 Neonatal Programming... 3
1.2.1 Neonatal programming: preconceptual, periconceptual and preimplantation periods………...4
1.2.2 Neonatal programming: uterine period ... 5
1.2.3 Neonatal programming: postnatal period ... 6
1.3 Metabolic syndrome ... 6
1.3.1 Definitions of metabolic syndrome ... 7
1.3.2 Metabolic syndrome and the dual burden of disease ... 8
1.3.3 Metabolic syndrome and fructose... 9
1.3.3.1 Fructose metabolism ... 10
1.4 Treatment of metabolic syndrome ... 12
1.4.1 Ursolic acid ... 13
1.4.1.1 Anti-hyperglycaemic and anti-diabetic effects of ursolic acid ... 14
1.4.1.2 Anti-obesity and anti-hyperlipidaemic effects of ursolic acid ... 14
1.4.1.3 Hepatoprotective effects of ursolic acid... 15
1.4.1.4 Anti-inflammatory and anti-cancer effects of ursolic acid ... 15
1.4.1.5 Other pharmacological effects of ursolic acid ... 16
1.5 Justification of the study ... 16
1.6 Aim of the study ... 17
x
CHAPTER 2 : MATERIALS AND METHODS ... 19
2.1 Ethical clearance for the study ... 20
2.2 Housing and general care of the animals ... 20
2.3 Chemicals and reagents used ... 20
2.4 Study design... 21
2.5 Measurement of body mass ... 23
2.6 Oral glucose tolerance tests ... 23
2.7 Terminal procedures ... 24
2.7.1 Tissue harvesting ... 24
2.8 Determination of hepatic lipid content ... 24
2.9 Clinical biochemistry assays... 25
2.10 Determination of bone linear growth and estimation of bone density ... 25
2.11 Statistical analysis ... 26
CHAPTER 3 : RESULTS ... 27
3.1 Effect of neonatal intake of ursolic acid on growth performance ... 28
3.1.1 Body mass measurements ... 28
3.1.2 Linear growth ... 31
3.2 Effect of neonatal intake of ursolic acid on the development of metabolic dysfunction ... 35
3.2.1 Circulating metabolites ... 35
3.2.2 Tolerance to an oral glucose load in adulthood ... 39
3.2.3 Food and fluid intake in adulthood ... 44
3.2.4 Adiposity ... 49
3.2.5 Hepatic storage of lipids ... 51
3.3 Effect of neonatal intake of ursolic acid on the morphometry of the gastrointestinal tract and accessory organs ... 54
3.3.1 Gastrointestinal tract (GIT) organs ... 54
3.3.2 Accessory organs ... 57
3.4 Effect of neonatal intake of ursolic acid on the general health profile ... 60
3.4.1 Surrogate markers of liver function ... 60
3.4.2 Surrogate markers of renal function in adulthood ... 62
3.4.3 Clinical biochemistry ... 64
CHAPTER 4 : DISCUSSION ... 66
4.1 Growth performance ... 67
xi
4.1.2 Linear growth ... 68
4.2 The development of metabolic dysfunction ... 69
4.2.1 Circulating metabolites ... 69
4.2.2 Tolerance to an oral glucose load ... 70
4.2.3 Food and fluid intake ... 71
4.2.4 Adiposity ... 72
4.2.5 Hepatic storage of lipids ... 72
4.3 Morphometry of the GIT and accessory organs ... 74
4.3.1 Gastrointestinal tract organs ... 74
4.3.2 Accessory organs ... 74
4.4 General health profile ... 75
4.4.1 Surrogate markers of liver function ... 75
4.4.2 Surrogate markers of kidney function ... 75
4.4.3 Clinical biochemistry ... 76
CHAPTER 5 : CONCLUSION AND RECOMMENDATIONS ... 78
5.1 Conclusion ... 79
5.2 Limitations and recommendations ... 79
CHAPTER 6 : REFERENCES ... 82
xii LIST OF FIGURES
Figure 1.1:Fructose and glucose metabolism in the liver ... 11 Figure 2.1: A diagrammatic representation of the study design. ... 22 Figure 3.1: Induction, weaning and terminal masses of male (A) and female (B) rats given different treatments. ... 29 Figure 3.2: Representative radiograph images of femora and tibiae of male rats. ... 33 Figure 3.3: Representative radiograph images of femora and tibiae of female rats. ... 34 Figure 3.4: Effect of ursolic acid on glucose tolerance in male (A) and female (B) rats. ... 40 Figure 3.5: Effect of ursolic acid on the total area under the curve of oral glucose tolerance test in male (A) and female (B) rats. ... 42 Figure 3.6: Hepatic lipid content in male (A) and female (B) rats in adulthood ... 52
xiii LIST OF TABLES
Table 1.1: Definitions of metabolic syndrome ...7
Table 3.1: Effect of ursolic acid on tibial and femoral masses, lengths and Seedor indices in male and female rats ... 32
Table 3.2: Effect of ursolic acid on circulating metabolites; fasting blood glucose and triglyceride concentrations (P35) ... 36
Table 3.3:Effect of ursolic acid on circulating metabolites in male and female rats in adulthood ... 38
Table 3.4: Food intake in male and female rats in adulthood ... 45
Table 3.5: Fluid intake in male and female rats in adulthood ... 47
Table 3.6: Effect of ursolic acid on adiposity... 50
Table 3.7: Effect of ursolic acid on the masses and lengths of GIT organs ... 55
Table 3.8: Effect of ursolic acid on masses of GIT accessory organs ... 58
Table 3.9: Effect of ursolic acid on surrogate markers of liver function ... 61
Table 3.10: Effect of ursolic acid on surrogate markers for renal function in male and female rats ... 63
xiv LIST OF ABBREVIATIONS
Acetyl Co-A: Acetyl coenzyme A AD: Alzheimer’s disease ADP: Adenosine diphosphate
AESC: Animal Ethics Screening Committee AIDS: Acquired Immune Deficiency Syndrome ALB: Albumin
ALP: Alkaline phosphatase ALT: Alanine aminotransferase AMP: Adenosine monophosphate AMPK: AMP-activated kinase AMY: Amylase
ANOVA: Analysis of variance AST: Aspartate aminotransferase ATP III: Adult treatment panel III ATP: Adenosine triphosphate BUN: Blood urea nitrogen CAS: Central animal services CO2: Carbon dioxide
DMSO: Dimethylsulphoxide
FAO: Food and Agriculture Organisation FW: Fructose water
GIT: Gastrointestinal tract GLUT: Glucose transporter HDL: High density lipoprotein HFCS: High fructose corn syrup
HIV: Human Immunodeficiency Virus IDF: International Diabetes Federation IUGR: Intrauterine growth restriction LDL: Low density lipoprotein LI: Large intestines
LKB1: Liver kinase binding -1 MS: Metabolic syndrome
xv NCD: Non- communicable disease
NCEP: National cholesterol education program NF-κB: Nuclear factor kappa-B
OGTT: Oral glucose tolerance test PKA: Protein kinase A
PPAR-α: Peroxisome proliferator activated receptor-alpha PPAR-γ: Peroxisome proliferator activated receptor-gamma PW: Plain water
ROS: Reactive oxygen species rTL: Relative to tibial length SI: Small intestines
TBIL: Total bilirubin TP: Total protein UA: Ursolic acid
UHC: Universal Health Coverage VLDL: Very low density lipoprotein w/v: weight/volume
WHA: World Health Assembly WHO: World Health Organisation
1
2 1.1 Dissertation structure and study background 1.1.1 Dissertation structure
This dissertation is comprised of six chapters; introduction, materials and methods, results, discussion, conclusions and recommendations and references. The introduction includes the literature review; exploring the concepts of neonatal programming, metabolic
syndrome and the pharmacology of ursolic acid. The justification of the study, aims of the study and the hypotheses used are also included in the introduction. The materials and methods used in the study, results obtained, discussion and recommendations arising from the study follow in subsequent chapters.
1.1.2 Background
The indiscriminatory increase in metabolic syndrome (MS), characterised by several metabolic abnormalities, in children and adults worldwide is alarming (Harris, 2013, Ahima et al., 2016). Genetic factors have been implicated in the aetiology of the syndrome as epidemiological and animal studies have shown transgenerational and multigenerational links (Heindel et al., 2015, Abou and Mani, 2016). The rate at which the syndrome is spreading, however, defies a solely genetic cause (Li et al., 2011, Lillycrop and Burdge, 2011, Vickers, 2011). Therefore, the contribution of environmental factors (nutrition, exercise and early life conditions) is under the spotlight (Gadgil et al., 2015, Padmanabhan et al., 2016, Xiao et al., 2016).
As the pathogenesis of the syndrome is yet to be fully elucidated and the syndrome is multifactorial, there is no specific treatment available (Bruce and Byrne, 2009, Mahajan et al., 2010). Current interventions have resulted in the discontinued use of some
pharmaceutical agents whilst marginal success rates have accompanied lifestyle changes (Giugliano et al., 2008, Kaur, 2014). Consequently, focus has shifted to the potential of phytochemicals abundant in fruits, herbs and vegetables (Graf et al., 2010, Akaberi and Hosseinzadeh, 2016). Pentacyclic triterpenes of the ursane group, such as ursolic acid (UA), have been shown to ameliorate metabolic syndrome associated abnormalities in adults (Rao et al., 2011, Sundaresan et al., 2012, Li et al., 2014). However, no work has been done to show the potential preventive effects of UA, if administered during the critical periods of sensitivity, against the development of metabolic syndrome later in life.
3
This study, therefore, investigated the effects of UA on early programming of subsequent diet-induced metabolic dysfunction in adulthood.
1.2 Neonatal Programming
Neonatal programming is a phenomenon that explains how the environment experienced in early life can have a long-term effect on the physiology, metabolism and therefore health of an individual (Martin-Gronert and Ozanne, 2012). The pre-conception, uterine and immediate postnatal periods are critical windows of physiological sensitivity (Aiken and Ozanne, 2013a, Wells, 2014). Environmental perturbations during these critical periods can trigger changes in organ and system development and if the changes are permanent, programming results (Gluckman et al., 2005, Barker, 2007, Cota and Allen, 2010). The foetus and neonate reprogram to favour early survival and improve success in an expected postnatal environment (Gluckman et al., 2008). A mismatch between the expected
postnatal environment and the prevailing postnatal environment, however, can increase susceptibility to disease later in life (Cagampang et al., 2011, Velkoska and Morris, 2011, Reynolds et al., 2015).
Epidemiological and animal studies have shown that the outcomes of the early
programming depend on the timing, duration, type and severity of insult and are at times sex-specific (Rinaudo and Wang, 2012, Aiken and Ozanne, 2013a, Goran et al., 2014). Changes in organ morphology and function programmed prenatally can be ameliorated or exacerbated postnatally (Ross and Desai, 2005). This is because neonatal programming can have either adverse or beneficial effects to disease later in life (Koletzko et al., 2011, Zohdi et al., 2012, Lewis et al., 2014). For instance, male rat pups receiving leptin
supplementation during lactation then a high fat diet post-weaning were found to have lower body mass, fat accumulation and feed intake preventing metabolic dysfunction later in life (Pico et al., 2007, Priego et al., 2010). In addition, the consequences of an
early/perinatal intervention can appear either spontaneously or they can be induced by another intervention after a period of latency (Heindel et al., 2015, Sun et al., 2015). The latter phenomenon is recognised in the “two hit” hypothesis for disease (Knudson, 1971, Day and James, 1998).
The two hit hypothesis states that a primary intervention (“first hit”) may sensitise an organ and lead to physiological alterations (Knudson, 1971, Erdélyi et al., 2013, Morris et al., 2015). The alterations may be immediately expressed leading to organ malfunction and
4
ultimately disease or may be suppressed (Bayer et al., 1999, Lahiri et al., 2007, Lahiri et al., 2009, Heindel et al., 2015). A second intervention (“second hit”), however, may unmask the suppressed effects leading to disease or amplify the effects of the “first hit” (Tsukamoto et al., 2009, Howard, 2013). For example, in the progression of non-alcoholic fatty liver disease (NAFLD), steatosis as a result of insulin resistance and/or obesity is usually the “first hit” (Dowman et al., 2010, Fabbrini et al., 2010, Tiniakos et al., 2010). Steatosis makes the liver vulnerable to a number of “second hits” including inflammatory cytokines, mitochondrial dysfunction and gut microbiota which ultimately lead to
steatohepatitis and fibrosis (Tilg and Moschen, 2010, Pais et al., 2011, Lau et al., 2015). In studies using sheep, maternal obesity in the periconceptual period (“first hit”) has been found to increase fat mass (Rattanatray et al., 2010), alter hepatic lipid metabolism (Nicholas et al., 2014) and insulin signalling (Nicholas et al., 2013) in the offspring. Maternal obesity in late gestation (“second hit”) results in the increased expression of lipogenic and adipogenic genes in the offspring (Muhlhausler et al., 2007, Long et al., 2015).
1.2.1 Neonatal programming: preconceptual, periconceptual and preimplantation periods
The maternal health status is important before, during and after conception as it has lasting metabolic effects on the offspring (Cardozo et al., 2011, Smith and Ryckman, 2015). In the preconception period, maternal metabolic syndrome alters the biochemical composition of follicular fluid, adversely affecting oocyte quality predisposing the resulting offspring to metabolic abnormalities (Leroy et al., 2012, Gu et al., 2015, Hou et al., 2016). Maternal over- and undernutrition result in epigenetic modifications which adversely alter oocyte maturity, blastocyst development and prenatal survival during the periconceptual and preimplantation periods (Robker, 2008, Ashworth et al., 2009, Niakan et al., 2012).
Changes during these periods can alter whole cell lineages (Burdge et al., 2011, Aiken and Ozanne, 2013b). The extent to which the placenta can mitigate or exacerbate these
changes, however, is unknown (Godfrey, 2002, Fowden et al., 2006). Undernutrition in these periods has been shown to increase susceptibility to cardiovascular anomalies including hypertension, small heart mass, excess in angiotensin converting enzyme and altered arterial vasodilation (Ashworth et al., 2005, Sinclair et al., 2007, Watkins et al., 2008, Watkins et al., 2010). Maternal overnutrition and obesity alter cardiovascular and behavioural mechanisms evidenced by distorted arterial contraction and dilation, pulse
5
pressure and stress responses in offspring (Gardner et al., 2004, Torrens et al., 2009, Hernandez et al., 2010). During embryonic development, maternal obesity and insulin resistance alter various metabolic pathways predisposing offspring to insulin resistance, hypercholesterolaemia and hyperleptinaemia in adulthood (Doblado and Moley, 2007, Hwang et al., 2010, Cardozo et al., 2011). Studies during this period have mostly been done in animals but are also important in the context of human in vitro reproduction where cardio-metabolic dysfunction is evident (Bertram and Hanson, 2001, Ceelen et al., 2008, Odom and Segars, 2010).
1.2.2 Neonatal programming: uterine period
Both maternal under and over-nutrition have adverse programming effects in utero. The uterine period is marked by vast gametogenesis as well as organogenesis such that nutritional manipulations cause permanent structural and reproductive alterations which affect functionality (Rhind et al., 2001). For example, in both altricial species and humans, the islets of the pancreas develop during gestation (Robb, 1961, Bouwens et al., 1997, Fowden and Hill, 2001). They then undergo remodelling during the first 2-3 postnatal weeks in altricial species and up to 4 years of age in humans (Robb, 1961, Hellerström and Swenne, 1991, Hill et al., 2000). Poor nutrition during these periods can alter islet
morphology and function which may lead to increased susceptibility to diabetes in adulthood (Dahri et al., 1995, Fowden and Hill, 2001, McMillen and Robinson, 2005). In under-nourished mothers, intrauterine growth restriction (IUGR) is a foetal coping mechanism which has been associated with epigenetic changes (Thompson et al., 2010, Xu et al., 2013). These changes lead to low birth weight babies who are likely to develop impaired glucose tolerance (Yajnik and Deshmukh, 2008), type-2 diabetes
(Kanaka‐Gantenbein, 2010), hypertension (Hinchliffe et al., 1992, Mackenzie and Brenner, 1995) and an increased risk of coronary heart disease (Eriksson et al., 2000) in adulthood. The loss of structural and functional units such as nephrons, cardiomyocytes and
pancreatic cells due to IUGR may be instrumental in the metabolic complications
experienced later in life (McMillen and Robinson, 2005, Dumortier et al., 2007, Zohdi et al., 2012).
One would expect that if the mother eats an obesogenic diet during pregnancy then the growing foetus reprograms and does not suffer from ill health after exposure to an obesogenic diet later in life. Studies, however, have shown that maternal overnutrition
6
leads to obesity and cardiovascular disorders in the offspring later in life (Li et al., 2011, Simmons, 2011, Brenseke et al., 2013). Obesity has been linked to leptin resistance and increased orexigenic neuropeptide production as foetal neural pathways, particularly those affecting feed intake, are reprogrammed in response to maternal overnutrition (Howie et al., 2009). Adipogenesis takes place during the late gestational phase and during the early postnatal period (Widdowson, 1968). Maternal overnutrition promotes fat accumulation in foetus which predisposes the offspring to obesity later in life (Samuelsson et al., 2008, Shankar et al., 2008). Excess circulating maternal lipids have also been associated with increasing the susceptibility of non-alcoholic fatty liver disease in offspring (Oben et al., 2010, Stewart et al., 2013).
1.2.3 Neonatal programming: postnatal period
In some mammals, organ development is incomplete at birth and continues in the early postnatal period (Patel and Srinivasan, 2011, Moore et al., 2015). Nutritional insults encountered during this suckling period can act as triggers for the induction of lasting programming effects (Guilloteau et al., 2009, Langley‐Evans, 2009, Portha et al., 2011). Over-nutrition during the postnatal period is thought to program central appetite regulators as well as glucose and lipid metabolism increasing the risk of metabolic dysfunction in adulthood (Chen et al., 2009). Thus, an altered milieu in the immediate postnatal period with metabolic perturbations may cause epigenetic modifications resulting in changes in gene promoter region methylation thereby affecting gene expression in pathways
associated with a range of physiologic processes (Jirtle and Skinner, 2007, Simmons, 2011). The phenotypic effects of these epigenetic modifications may not manifest until later in life, especially if they affect genes modulating responses to later environmental challenges such as dietary change (Gicquel et al., 2008, Szyf, 2009). Neonatal
programming can be a result of interactions between various environmental stressors which can lead to increased susceptibility to some similar diseases (Heindel et al., 2015). It has therefore been implicated in the aetiology of the metabolic syndrome.
1.3 Metabolic syndrome
The metabolic syndrome is a cluster of cardiovascular risk determinants, including
abdominal adiposity, glucose intolerance, hypertriglyceridaemia, non-alcoholic fatty liver disease (NAFLD) and decreased high density lipoprotein cholesterol (Alberti and Zimmet, 1998, National Cholesterol Education Program, 2002, Nomura and Yamanouchi, 2012).
7
Depending on sex, race, age, the population studied, setting (rural or urban area) and the definition of the syndrome used, global prevalence of metabolic syndrome can range from as low as less than 10% to as high as above 60% (Erasmus et al., 2012, Kaduka et al., 2012, Bhat et al., 2015).
1.3.1 Definitions of metabolic syndrome
Including those for children and adolescents, as many as 40 definitions exist for metabolic syndrome with the three most widely used shown below in Table 1.1 (Ford and Li, 2008, Kassi et al., 2011, Thaman and Arora, 2013). Being a public health concern, the many definitions have led to confusion in the identification, diagnosis and quantification of individuals at risk and sufferers of the syndrome (Alberti and Zimmet, 1998, Blaha and Elasy, 2006).
Table 1.1: Definitions of metabolic syndrome World Health Organisation
(WHO)
International Diabetes Federation (IDF)
National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III)
Emphasises the presence of
insulin resistance and any two of the following: Obesity
Hypertension
High triglyceride levels Reduced high density
lipoprotein cholesterol (HDL)
Microalbuminuria (Alberti and Zimmet, 1998)
Emphasises the presence of
central obesity (waist circumference with ethnicity specific values) plus any two of the following:
Raised triglycerides Raised blood pressure Reduced HDL Raised fasting plasma
glucose
(International Diabetes Federation, 2005 )
Any three of the
following:
Abdominal obesity Elevated triglycerides Reduced HDL Raised blood pressure Raised fasting glucose (National Cholesterol Education Program, 2002)
Currently, a harmonised criteria is used to reduce disparities in research and to establish the incidence and prevalence of MS worldwide (Alberti et al., 2009). In tandem with the
8
NCEP definition, it emphasises that there be no mandatory component and that any three out of five abnormal components be considered as MS. However, it was agreed that waist circumference (with ethnicity specific values) be used as a preliminary screening tool (Alberti et al., 2009).
1.3.2 Metabolic syndrome and the dual burden of disease
Traditionally, the cardio-metabolic risk factors of MS have been known to be widespread in developed nations whilst communicable diseases and undernutrition have mostly affected developing nations (Horton, 2007, Miranda et al., 2008, Ellulu et al., 2014). This has been partly due to the different nutritional and lifestyle trends. Globalisation, however, has and continues to bring a number of transitions in its wake: nutritional, epidemiological and health (Popkin, 2002, Popkin et al., 2012, Azuike et al., 2013). The nutrition transition promoted the global availability of cheap sugar (high corn fructose syrup), vegetable oils and fats resulting in direct competition with grain products (Drewnowski and Popkin, 1997, Chopra et al., 2002, Hawkes, 2005). Consequently, there have been changes in nutrition and disease trends.
In developing nations, a decline in coarse grain consumption has fuelled increased consumption of refined grains and calorie-rich food whilst fruit and vegetable intake remain inadequate (Popkin and Ng, 2007, Kearney, 2010, Khan and Talukder, 2013). This has concomitantly led to the epidemiological and health transitions in which MS and other non-communicable diseases (NCDs) have been on the rise (Popkin et al., 2012). In some countries, NCDs surpass mortality of infectious diseases like HIV/AIDS (Kelly and Fuster, 2010). Globalisation also gives rise to urbanisation which paves way for sedentary
lifestyles which worsen MS (Ruel et al., 2008).
The nutrition transition promotes undernutrition in the form of micronutrient deficiency. Micronutrient deficiency weakens natural immunity which increases the susceptibility to communicable diseases exacerbating mortality rates (Katona and Katona-Apte, 2008, França et al., 2009, Singer, 2013).Despite agricultural technological advances, some countries in Africa, particularly war-torn nations (South Sudan and Democratic Republic of Congo) and those suffering from periodic droughts (parts of Ethiopia, Kenya, Uganda and Somalia, Eritrea), undernutrition in the form of macronutrient deficiency is still a problem (World Health Organisation, 2007, Louis and Hess, 2008, Bain et al., 2014).As a
9
result, most developing countries suffer the double burden of disease; both communicable and non-communicable diseases at household, community and population levels (Popkin, 2003, Kennedy et al., 2006).Most of these nations already have frail healthcare policies and systems and disintegrating social service structures (Sikosana et al., 1997, Bradshaw and Ndegwa, 2000, Foley, 2009). The emergence and contribution of MS to the burden of disease in developing countries therefore threatens to overwhelm the health sector (Evans, 2009, Bloom et al., 2012).
1.3.3 Metabolic syndrome and fructose
With the increase in fructose consumption coinciding with increases in metabolic
irregularities, fructose has been implicated in the genesis and progression of MS (Douard and Ferraris, 2008, Khitan and Kim, 2013). Fructose is the natural sugar found in honey and in many fruits (Shils and Shike, 2006). It is also a constituent of table sugar, sucrose (Harvey and Ferrier, 2011). Sweetness values are a measure of the relative sweetness of a substance with the standard being sucrose with a value of 100 (Shallenberger and Acree, 1971). Crystalline fructose has a sweetness value of 175 making it the sweetest natural sugar (Shallenberger and Acree, 1971, Shallenberger, 2012). The commercial hydrolysis of maize/corn yields glucose which is enzymatically isomerised to give fructose in varying glucose:fructose ratios in the production of high fructose corn syrup (HFCS) (Considine, 2012, Marie and Piggott, 2013). It is used as an ingredient in sweetened beverages and many processed foods as it is a cheap source of sugar with long shelf life and
moisterisation benefits (Hanover and White, 1993, Berdanier et al., 2007, Tappy and Lê, 2010).
The consumption of commercially produced fructose is increasing whilst natural fructose consumption is decreasing or remaining constant globally (Marriott et al., 2009). Being sweet, fructose is highly palatable and is thus favoured by consumers resulting in overfeeding (Tappy and Lê, 2010, Gibney et al., 2013). High fructose consumption, however, leads to metabolic dysfunction; increased concentrations of plasma free fatty acids, leptin, triglycerides, uric acid and abdominal adiposity (Hallfrisch, 1990, Melanson et al., 2008, Alzamendi et al., 2009). Impaired insulin sensitivity also results (Aeberli et al., 2013, Khitan and Kim, 2013). In a bid to curb the detrimental metabolic effects of sugars such as fructose, “sugar-tax” has been introduced in countries like Mexico
10
beverages) (Escobar et al., 2013, Cornelsen and Carreido, 2015). Many countries like South Africa are in the process of implementing the same policy (National Department of Health, 2012). Among all the monosaccharides, fructose has received widespread attention mainly due to the way it is metabolised in the body.
1.3.3.1 Fructose metabolism
The metabolic pathways of fructose and glucose in the liver are shown in Figure 1.1 overleaf. At low concentrations in the small intestine lumen, fructose is converted to glucose (Mayes, 1993, Douard and Ferraris, 2013). At high concentrations, however, it is absorbed and metabolised in the liver resulting in very small amounts of fructose (about 0.01 mmol/L) entering systemic circulation (Mayes, 1993, Bray, 2007). Non-insulin dependent glucose transporters, GLUT-5 and GLUT-2, facilitate the movement of fructose into intestinal cells and into the liver respectively (Keembiyehetty et al., 2006, Manolescu et al., 2007, Thorens and Mueckler, 2010). The testes, spermatozoa, kidneys, adipose tissue, muscle and to a lesser extent the brain express GLUT- 5 (Gropper and Smith, 2012, Roy and Krishna, 2013). In cells, fructose is converted to fructose-1-phosphate by the enzyme fructokinase (Ishimoto et al., 2012). Through catalysis by aldolase B, fructose-1-phosphate is then converted to the trioses: glyceraldehyde-3-fructose-1-phosphate and
dihydroxyacetone phosphate, which are the precursors for phospholipid and triacylglycerol synthesis (Harvey and Ferrier, 2011, Seidler, 2013).
Glucose, in comparison, is transported into cells by an insulin dependent transporter, GLUT-4 (Huang and Czech, 2007, Douard and Ferraris, 2013). It is converted to glucose-6-phosphate by hexokinase then to the triose phosphates and finally into pyruvate during glycolysis (Harvey and Ferrier, 2011, Bender, 2014). Glucose metabolism is highly regulated by the energy status of the cell through negative feedback mechanisms
(Lieberman et al., 2006, Gropper and Smith, 2012). Phosphofructokinase, a rate limiting enzyme, catalyses the formation of fructose-1,6-bisphosphate (precursor of the triose phosphates) and is inhibited by citrate and adenosine triphosphate (Berg et al., 2002, Bender, 2014). The formation of triose phosphates in fructose metabolism, however, is void of this regulatory step thus it is a source of abundant and unregulated carbon atoms (Elliott et al., 2002, Rippe, 2014).
11
Figure 1.1:Fructose and glucose metabolism in the liver (Tappy and Lê, 2010). Abbreviations: -P; Phosphate, Acetyl Co-A; Acetyl coenzyme A, CO2; carbon dioxide,
ATP; Adenosine triphosphate, ADP; Adenosine diphosphate, AMP; Adenosine monophosphate GLUT 2; glucose transporter 2.
Fructose facilitates de novo hepatic lipogenesis, lactate production, glycogen synthesis and gluconeogenesis (Park et al., 1992, Shils and Shike, 2006). This puts it at the crux of the progression of MS as accumulation of hepatic lipids contributes to dyslipidaemia, NAFLD and tissue-specific insulin resistance (Birkenfeld and Shulman, 2014, Perry et al., 2014). Furthermore, overconsumption as a result of fructose feeding contributes to obesity (Bray et al., 2004, Brown et al., 2008). Fructose metabolism also results in the depletion of hepatic adenosine triphosphate (ATP), which results in the production of adenosine monophosphate (AMP) and uric acid fuelling hyperuricaemia (Van den Berghe, 1985, Hallfrisch, 1990, Johnson et al., 2013). Hyperuricaemia has been implicated in the pathogenesis of insulin resistance leading to diabetes and hypertension (Gustafsson and Unwin, 2013, Johnson et al., 2013, Soltani et al., 2013). Putative mechanisms include endothelial dysfunction through reduction of nitric oxide, induction of the renin
12
angiotensin system and smooth muscle cell proliferation (Khosla et al., 2005, Sanchez-Lozada et al., 2005, Corry et al., 2008).
1.4 Treatment of metabolic syndrome
There is no specific treatment for MS (Reddy and Rao, 2006, Giugliano et al., 2008). As such, lifestyle modifications and pharmaceutical agents are being used to manage MS (Marvasti and Adeli, 2010, Brenseke et al., 2013). Lifestyle changes such as body mass reduction and increased physical activity are normally the first-line of therapy in the management of MS (Ricanati et al., 2011, De Lorgeril, 2012). This is because they can improve all facets of the syndrome (Dalle Grave et al., 2010, Yamaoka and Tango, 2012, Hartley, 2014). With increasing severity of MS, however, pharmaceutical agents are used together with lifestyle modifications (Rubio-Ruiz et al., 2013).
Lipid lowering drugs which reduce low density lipoprotein cholesterol (statins) and increase high density lipoprotein cholesterol (niacin and fibrates) have been used to
manage MS (Michos et al., 2012, Elkes, 2016). Insulin sensitising drugs such as metformin and peroxisome proliferator activated receptor gamma (PPAR-γ) agonists with
anti-diabetic properties (thiazolidinediones) are also used to reduce blood glucose in the
management of MS (Derosa and Maffioli, 2010, Rojas and Gomes, 2013, Song, 2016). The undesirable side effects, low efficacy and high cost associated with the use of
pharmaceutical agents have made them inaccessible and have led to their discontinued use leaving few drugs that can be used (Elangbam, 2009, Rodgers et al., 2012). This is a hindrance to one of WHO’s overarching goals of universal health coverage (UHC). The UHC “seeks to ensure that all people have access to promotive, preventive, curative and rehabilitative health services, of sufficient quality to be effective, while also ensuring that they do not suffer financial hardship when paying for these services” (WHO, 2015). With WHO predicting that deaths from NCDs such as MS will increase from 38 million to 52 million by 2030, there has been an increased use of traditional medicine such as
ethnomedicines (Eddouks et al., 2012, WHO, 2014, WHO, 2015). The increased use of ethnomedicines was evident in Korea where annual expenditure on traditional medicine rose from US$ 4.4 billion in 2004 to US$ 7.4 billion in 2009 (Johnson, 2016). This trend was also apparent globally as an estimated US$83 billion was spent on traditional medicine in 2008 and an exponential rate of increase observed (Robinson and Zhang, 2011).
13
and readily available than conventional medicine (Cooper and Yamaguchi, 2013, WHO, 2015). In most developing countries ethnomedicines provide the bulk of primary health care (Payyappallimana, 2010, WHO, 2015). Additionally, traditional medicine is also being widely used in developed countries with 70% of Canadians, 82% of Australians and over 100 million users in Europe (Johnson, 2016). The World Health Assembly (WHA) and WHO have therefore put in place strategies to facilitate the safe use, regulation and promotion of ethnomedicines (World Health Assembly, 2009, WHO, 2015). These strategies have facilitated the integration of traditional and complementary medicine into the health system in countries such as China, Korea and Switzerland (Government of China, 2010, Swiss Confederation, 2011, Frass et al., 2012).
With regards to MS,researchers are currently investigating the therapeutic potential of naturally occurring phytochemicals which are abundant in fruits and vegetables (Kim et al., 2011, Holubková et al., 2012). Many of the phytochemicals display pharmacological and biochemical effects that include inhibition of several different enzyme systems related to absorption and metabolism of carbohydrates and lipids (Yoshizumi et al., 2006, de Melo et al., 2010, Gupta and Prakash, 2014). Pentacyclic triterpenes belonging to the lupane, oleanane and ursane series are some of the phytochemicals being explored (Hasani-Ranjbar et al., 2009, Alqahtani et al., 2013, Castellano et al., 2013). As most of these phytochemicals are taken orally, their interaction with the gastrointestinal tract (GIT) is of great importance (Gavhane and Yadav, 2012, McClements et al., 2015). Studies have shown that although development of the GIT is pre-programmed, nutritional manipulation in the intrauterine and early postnatal periods can affect it resulting in precocious
maturation leading to higher body mass gains and enhanced bone development (Linderoth et al., 2005, Puzio et al., 2007). Ursolic acid is one of the phytochemicals being explored in the management and treatment of MS (Li et al., 2014, Nazaruk and Borzym-Kluczyk, 2014).
1.4.1 Ursolic acid
Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) is an ursane pentacyclic triterpenoid that exists naturally in plants as a free acid or aglycone (Liu, 1995, Sun et al., 2006). It has been isolated from a number of fruits including apples (He and Liu, 2007), guavas (Begum et al., 2004), loquats (Zhou et al., 2007) and olives (Somova et al., 2003). It has also been isolated from medicinal herbs such as rosemary (Huang et al., 1994), basil (Chiang et al.,
14
2005), sage (Le Men and Pourrat, 1952) and thyme (Ismaili et al., 2001). Being biologically active both topically and internally, UA exhibits a wide range of pharmacological effects.
1.4.1.1 Anti-hyperglycaemic and anti-diabetic effects of ursolic acid
Diabetes mellitus is a result of altered insulin secretion and/or insulin action which perturbs fat, protein and carbohydrate metabolism resulting in chronic hyperglycaemia (Alberti and Zimmet, 1998). Lowering postprandial blood glucose concentration is one of the therapeutic strategies employed in managing diabetes mellitus (Nathan et al., 2009, Nyenwe et al., 2011). Ursolic acid extracted from Cornus officinalis Sieb. et Zucc (Gao et al., 2008) and Osmanthus fragrans (Kang et al., 2012) was found to lower fasting blood glucose and postprandial glucose concentrations in diabetic rats.
1.4.1.2 Anti-obesity and anti-hyperlipidaemic effects of ursolic acid
Adipocytes secrete hormones (leptin and adiponectin), store lipids and are insulin sensitive (Camp et al., 2002, Havel, 2004). Alterations in adipocyte metabolism have adverse
effects, with obesity being marked by hyperplasia and hypertrophy of adipocytes (Drolet et al., 2008, Jo et al., 2009, Stephens, 2012). Ursolic acid stimulates lipolysis in mature 3T3-L1 adipocytes through a cyclic-AMP dependent protein kinase A (PKA) pathway (Li et al., 2010). Furthermore, UA suppresses pre-adipocyte differentiation and adipogenesis through the liver kinase binding-1/AMP-activated protein kinase (LKB1/AMPK) pathway in 3T3-L1 adipocytes, exhibiting anti-obesity properties (He et al., 2013).
Dietary fat is thought to promote body fat storage more effectively than dietary
carbohydrates leading to metabolic anomalies (Consultation, 1998, Brown et al., 2008). However, fat absorption is only possible after fat digestion by lipase (Gargouri et al., 1997). Following oral administration of lipid emulsion in rats, UA has been shown to have lipase-inhibiting properties which prevent increases in plasma triacylglycerol
concentrations (Kim et al., 2009). In non-obese mice, UA promotes thermogenesis, energy expenditure increase of brown fat which aids obesity management strategies (Kunkel et al., 2012).
15 1.4.1.3 Hepatoprotective effects of ursolic acid
The liver is the body’s metabolic hub (Wall and Porter, 2014, Feldman et al., 2015). It is responsible for detoxification and the synthesis, metabolism, storage and redistribution of carbohydrates, proteins and lipids (Berg et al., 2002, Rui, 2014). Hypercaloric diets, sedentary lifestyles and drug use have deleterious effects on the liver resulting in NAFLD, cholestasis and liver injury (Epstein et al., 1998, Lee, 2003, Zelber-Sagi et al., 2007, Masarone et al., 2014). Thioacetamide (Akhtar and Sheikh, 2013), galactosamine (Keppler et al., 1968) and carbon tetrachloride (Hübner, 1965, Shi et al., 1998) are used to induce hepatotoxicity in rats as they decrease hepatocyte viability as well as disrupt bile secretion. Pre-treatment of rats with UA improves hepatocyte viability as well bile secretion in a similar manner as the hepatoprotective drug silymarin (Binduja et al., 1996, Feher and Lengyel, 2012). Ursolic acid ameliorates paracetamol-induced hepatotoxicity in rats exhibiting its anti-cholestatic and anti-choleretic effects in rats (Shukla et al., 1992). Thought to be the hepatic facet of MS, NAFLD encompasses steatosis, inflammation, hepatocellular injury and fibrosis ultimately progressing to liver cirrhosis and
hepatocellular carcinoma (Angulo, 2002, Dowman et al., 2010, Takahashi and Fukusato, 2014). Endoplasmic reticulum stress has been implicated in fuelling the progression of steatosis to more aggressive forms of NAFLD (Gentile et al., 2011, Lake et al., 2014). Li et al. (2015) administered UA to diabetic mice and L02 palmitic acid stimulated cells.
Hepatic steatosis, liver mass and concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were reduced in the diabetic rats whilst lipid accumulation was reduced in L02 cells. Through enhanced lipid β-oxidation and inhibition of
endoplasmic reticulum stress, UA exhibits hepatoprotective properties which may be crucial in NAFLD therapy.
1.4.1.4 Anti-inflammatory and anti-cancer effects of ursolic acid
Inflammation is a non-specific, adaptive immune response to any stimuli that cause cell injury (Markiewski and Lambris, 2007). In response to tissue damage, inflammation allows for repair of damaged tissue (Krafts, 2010) whilst in pathological conditions such as
cancer, chronic inflammation may result in tissue damage and organ dysfunction (Coleman and Tsongalis, 2009, Dubois, 2015). Ursolic acid exhibits its anti-inflammatory property through attenuation ofinducible nitric oxide synthase and cyclooxygenase-2 expression in the murine macrophage cell line,RAW264.7 (Suh et al., 1998, Ryu et al., 2000). Ursolic
16
acid also has anti-leukaemic and anti-cancer properties through its suppression of nuclear factor kappa-B (NF-κB) activation (Najid et al., 1992, Shishodia et al., 2003). The anti-cancer effect of UA has also been illustrated in lung anti-cancer (Kim et al., 2015), prostate cancer (Park et al., 2013), ovarian cancer (Wang et al., 2009), breast cancer (Gim et al., 2010) and hepatocellular carcinoma (Yang et al., 2010) cell lines through apoptosis. Ursolic acid has been used in cancer Phase I clinical trials (Zhu et al., 2013, Qian et al., 2015).
1.4.1.5 Other pharmacological effects of ursolic acid
Ursolic acid displays cardiotonic (Somova et al., 2004), anti-ulcer (Shih et al., 2004) and nephroprotective (Pai et al., 2012) properties in studies using rodents. Studies have
demonstrated UA’s anti-viral effects against human immunodeficiency virus (Quéré et al., 1996) and hepatitis C (Kong et al., 2013) in NS5B cells. Extracts from UA containing plants Satureja parvifolia, Morinda lucida and Mimusops caffra exhibited anti-malaria activity against chloroquine-sensitive Plasmodium falciparum strains (Baren et al., 2006, Cimanga et al., 2006, Simelane et al., 2013). In mice, oral administration of UA was found to reduce parasitic loads during the acute phase of Trypanosoma cruzi infections (da Silva Ferreira et al., 2013). In the nematodes that cause elephantiasis, Brugia malayi and
Wuchereria bancrofti, UA induced apoptosis by altering enzyme activity in worms isolated from infected people (Saini et al., 2014). Ursolic acid has also been shown to be
neuroprotective exhibiting anxiolytic (Pemminati et al., 2011), anti-nociceptive (González-Trujano et al., 2012, Verano et al., 2013) and anti-depressant like (Machado et al., 2012, Colla et al., 2014) properties in rodents.
1.5 Justification of the study
Most studies assessing the effects of UA on metabolic dysfunction used adult animals primarily and used UA as a treatment method (Jayaprakasam et al., 2006, Jang et al., 2009, Rao et al., 2011). Metabolic dysfunction, however, has the potential to be reversed by nutritional and therapeutic interventions during physiologically sensitive periods (Vickers, 2011). Additionally, the prevalence of MS seems to be sex-biased with females being more prone to the syndrome than males (Tonstad et al., 2007, Houti et al., 2016). With the manifestation of the metabolic abnormalities in different organs being sex-specific and with most studies having been done in males, there is need for sex-specific studies (Beigh and Jain, 2012, Tsai et al., 2014). Therefore, although UA displays great pharmacological
17
potential to ameliorate MS, there is a paucity of data of intervention strategies using UA during the period of developmental plasticity. As such, this study aimed to investigate the effects of UA on neonatal programming and to assess if it could protect against the
development of diet-induced metabolic dysfunction in adulthood in Sprague Dawley rats in a sex-dependent manner.
1.6 Aim of the study
The main aim of this study was to determine the effect of UA on neonatal programming of metabolic dysfunction in rats fed a high fructose diet and to assess its potential to protect against the subsequent development of metabolic syndrome in adulthood. The specific objectives of this study were to determine the effects of UA on;
1. neonatal programming of diet-induced metabolic dysfunction and the subsequent health outcomes in adulthood.
2. growth performance of rats by measuring; a. body mass gain.
b. linear growth (length, mass and density of the long bones: femur and tibia). 3. the development of metabolic dysfunction by measuring;
a. circulating levels of metabolites: glucose, cholesterol and triglycerides. b. tolerance to an oral glucose load.
c. food and fluid intake. d. adiposity.
e. hepatic storage of lipids.
4. the morphometry of the GIT (and accessory organs) by measuring lengths and masses of visceral organs.
5. the general health profile of the rats by measuring;
a. surrogate markers of liver function: alanine transaminase (ALT), alkaline phosphatase (ALP), total bilirubin and albumin.
b. surrogate markers of renal function: creatinine and blood urea nitrogen. c. clinical biochemistry: total protein, calcium, phosphate, amylase and
globulins.
1.7 Hypotheses
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H0: Ursolic acid has no effect on neonatal programming and does not protect against the subsequent development of metabolic dysfunction in Sprague Dawley rats fed a high fructose diet in the neonatal period and/or in adulthood. In addition, that no differences exist between male and female responses to UA treatment.
H1: Ursolic acid has an effect on neonatal programming and protects against the subsequent development of metabolic dysfunction in Sprague Dawley rats fed a high fructose diet in the neonatal period and/or in adulthood. In addition, that differences exist between male and female responses to UA treatment.
19
CHAPTER 2 : MATERIALS AND
METHODS
20 2.1 Ethical clearance for the study
All animal experiments were carried out according to protocols approved by the Animal Ethics Screening Committee (AESC) of the University of the Witwatersrand, AESC number 2014/49/D (see appendix for certificate).
2.2 Housing and general care of the animals
Sprague Dawley dams with suckling male and female rat pups from the Central Animal Services (CAS) at the University of the Witwatersrand were used in the study. Each dam, with a litter of 9-12 pups, was housed in perspex cages lined with wood shavings in temperature (24˚C±2˚C), light (12h light:12h dark; 0700:1900 clock time) and ventilation controlled rooms. A commercial rat chow (Epol®, Johannesburg, South Africa) and clean drinking water were provided ad libitum for the dams. After weaning, the weaned rats were housed individually as described above.
2.3 Chemicals and reagents used
Dimethylsulphoxide (DMSO; Sigma-Aldrich, France) was reconstituted in distilled water to a final concentration of 0.5% and used as vehicle. Ursolic acid (Sigma-Aldrich, France) was prepared by dissolving 2 mg of ursolic acid in 10 μl of DMSO and making up to 2 ml with distilled water. The UA was prepared in bulk, aliquoted to 2 ml portions and stored in microtubules (Eppendorf) at -20˚C until use. Fructose (Nature’s choice, South Africa) was used to induce metabolic dysfunction. According to the manufacturer’s product
information label, the nutritional composition of the fructose was 1680 kJ energy, 0 g protein, 0.2 mg/100 g sodium, 99.8 g/100 g carbohydrates, 0 g fat and 0 g fibre. Fructose (20% w/v) was prepared by dissolving 20 g of fructose in tap water and making it up to 100 ml. Fructose (50% w/v) was prepared by dissolving 50 g of fructose in distilled water and making it up to 100 ml. The commercial rat chow (Epol®, Johannesburg, South Africa) had the following nutritional composition: 170 g/kg protein, 25 g/kg fat, 70 g/kg fibre, 25 g/kg calcium, 6 g/kg phosphorus and 6.5 g/kg lysine. Glucose solution (50% w/v; Radchem, South Africa) was made by dissolving 5 g of glucose in distilled water and filling it up to the 10 ml mark. A drop of food colouring (no nutritional value) (Robertsons, Retailer Brands (Pty) Ltd, South Africa) was added to 5 l of the drinking fluids and used to distinguish the fluids from one another.
21 2.4 Study design
Figure 2.1 overleaf shows a diagrammatic representation of the study design which aimed to simulate a ‘one hit’ and ‘two hit’ interventional study. A total of one hundred and seven male and female suckling pups were used (from 11 litters; 9-12 pups per litter). In the first stage of the study (which was from postnatal day 6 (P6) to P20), the first nutritional intervention “first hit” was introduced to induce neonatal programming. The pups in each litter were randomly assigned to four main treatment groups, each with a minimum of 26 pups in each treatment group. The rat pups were uniquely identified by marking with colour coded, non-toxic permanent marker ink on their tails. The rat pups received an oral administration of one of the following treatments;
Group 1 (control): *0.5 % DMSO (10 ml/kg b.w). The DMSO was used as a vehicle to dissolve the UA (Sundaresan et al., 2012) (n =27).
Group 2: *UA (10 mg/kg b.w) reconstituted in DMSO. We used this dose of UA because previous studies reported its effectiveness in reversing the symptoms of metabolic
syndrome (visceral adiposity, blood glucose concentrations and plasma lipids) in mice fed a high fat diet (Rao et al., 2011) (n =27).
Group 3: 50% fructose solution (10 ml/kg b.w) (n =27)
Group 4:*UA (10 mg/kg b.w) + 50% fructose solution (n =26). *= 10 ml/kg b.w
Orogastric administration of the treatments was done once daily between09:00 and 11:00 using an orogastric tube mounted on a 1 ml syringe.In-between gavaging, the pups were allowed to suckle freely on their respective dams.
In the second phase of the study (non-interventional phase), the pups were weaned on P21 and housed individually. They were fed a commercially supplied rat chow and supplied with clean plain drinking water ad libitum until P69. In order to assess if the interventions in the first phase had any early metabolic effects on the pups, metabolic assays were done on P35. After overnight fasting, blood was taken from a pin prick at the tip of alcohol-swab sterilised tails of the rats (Parasuraman et al., 2010). The blood was used to determine fasting glucose and triglyceride concentrations. A calibrated glucometer (Ascensia, Ireland) was used to determine blood glucose concentration whilst a calibrated Accutrend
22
triglyceride meter (Roche Diagnostics, Germany)was used to determine blood triglyceride concentration as per manufacturer’s instructions.
DMSO (n=27) UA (n=27) FR (n=27) FR+ UA (n=26) PW (n=14) 20% FW (n=13) N=107 Birth P6 P70 P126 1st Intervention 2nd Intervention P129 P21 Non-interventional period Treatment Days Termination OGTTs P35 Metabolic assays PW (n=14) 20% FW (n=13) PW (n=13) PW (n=14) 20% FW (n=14) 20% FW (n=12) Weaning
Figure 2.1: A diagrammatic representation of the study design. Abbreviations – DMSO; dimethylsulphoxide, UA; ursolic acid, FR; fructose, OGTTs; oral glucose tolerance tests, PW; plain drinking water and FW; fructose in drinking water, P; postnatal day.
The third phase of the study (second intervention; “second hit”) started on P70 until
termination (P129).Normal rat chow was given to all the rats, however, half the number of rats in each group were assigned to one of two treatments (with males and females in each group) wherein they received either plain drinking water or 20% fructose in drinking water.Administration of 20% fructose for eight weeks has been shown to induce obesity, dyslipidaemia, hyperglycaemia, hyperinsulinaemia and hypertension in rats (Barros et al., 2007, Mamikutty et al., 2014). Twice a week, the drinking bottles were washed and changed and fresh solutions were prepared and provided. A drop of food colouring was added to 500 ml of either plain drinking water or 20% fructose in drinking water. This was in order to differentiate the fluids from one another. During this phase of the study, food
23
and fluid intake were measured weekly using modification of Mamikutty et al. (2014) formulae:
Average daily food intake = [initial feed mass (g) – final feed mass (g)]/ number of days the feed was supplied.
Average daily fluid intake = [initial fluid volume (ml) – final fluid volume (ml)]/ number of days the fluid was supplied.
Both daily food and fluid intake were then expressed and reported as a percentage of body mass as g/ 100 g and ml/100g respectively (Ghezzi et al., 2012).
The feed was weighed using a balance (Snowrex Electronic Scale, Clover Scales,
Johannesburg) whilst a calibrated measuring cylinder was used to determine the volume of the fluids.
2.5 Measurement of body mass
In the first phase, the pups were weighed (Snowrex Electronic Scale, Clover Scales, Johannesburg) daily to ensure that the correct dosage ofthe various treatments was administered. Post-weaning, the rats were weighed twice every week in order to assess growth. The dams were also weighed twice every week as part of routine health checks. 2.6 Oral glucose tolerance tests
After eight weeks of intervention, tolerance to an oral glucose load was assessed on P126. The animals were habituated in perspex restrainers for an hour for three consecutive days prior to the procedure. The rats were fasted overnight but had ad libitum access to drinking water. Following sterilisation of the tail as described in Section 2.4, a fasting blood sample was taken following a pin-prick at the tip of the tail (time=0). Glucose (2 g/kg b.w) was then administered orogastrically and blood samples were collected from the tip of the tail after 15, 30, 60, 120 and 180 minutes to determine systemic glucose concentrations (Ghezzi et al., 2012). Blood glucose was determined using a calibrated glucometer
(Ascensia, Ireland) according to the manufacturer’s instructions. The rats were returned to their designated feeding regime for 48 hours prior to termination. The total area under the curve (AUC) was calculated from the OGTT results.
24 2.7 Terminal procedures
The rats were terminated on P129 after an overnight fast but had ad libitum access to drinking water. Fasting triglyceride concentrations were assessed using blood taken from a pin prick at the tip of the sterilised tail as previously described. A calibrated Accutrend triglyceride meter (Roche Diagnostics, Germany)was used to determine triglyceride concentration as per manufacturer’s instructions. The rats were euthanased using sodium pentobarbitone (200 mg/kg b.w).
2.7.1 Tissue harvesting
After euthanasia, the thorax was opened for organ harvesting and blood collection via cardiac puncture. Blood was collected into heparinised and plain tubes (BD Vacutainer, Plymouth, UK) and centrifuged (Rotofix 32A, Hettich Zentrifugen, Germany) at 4 000 G for 15 minutes. The supernatant (plasma/serum) was then pipetted into microtubes (Eppendorf) before being stored at -20˚C for further analysis. The heart, liver, stomach, pancreas, caecum, small and large intestines, visceral and epididymal fat were carefully dissected out. The stomach, small intestines, caecum and large intestine were weighed after being gently squeezed to remove any wastes. The lengths of large and small intestines were measured using a ruler mounted on a dissection board. The masses of visceral (and epididymal in males) fat pads, heart, pancreas, liver and kidneys were also measured using a balance (Presica 310M, Switzerland). The liver was stored at -20˚C for further analysis. All organ masses were corrected relative to tibial length by using the formula;
Organ mass relative to tibial length = organ mass (g) / length of tibia (mm) (Nunes-Souza et al., 2016).
2.8 Determination of hepatic lipid content
Determination of the liver lipid content was done by solvent extraction at the Agricultural Research Council (Irene Analytical Services Laboratory) using the Tecator Soxtec method (Official Methods of Analysis of Analytical Chemists, 2005). The liver samples were freeze-dried, milled and 1 g was placed into a pre-weighed extraction thimble. Fat-free cotton wool was used to plug the thimble before it was placed on a thimble holder.
Petroleum ether was added to the extraction cups before the cups were placed onto heating pads. The extraction process involved four stages: boiling (30 minutes), rinsing (30
25
minutes), petroleum ether recovery (10 minutes) and drying (30 minutes at 90 ± 5˚C). The extraction cups were then allowed to cool in a dessicator before the amount of the oil was determined using the following formula:
% fat = 100[(mass of cup plus fat – mass of cup) ÷ (mass of sample)] The test was done in triplicate.
2.9 Clinical biochemistry assays
An IDEXX VetTest Chemistry Analyser (IDEXX VetTest® Clinical Chemistry Analyser, IDEXX Laboratories Inc., USA) was used to measure the serum concentrations of alanine aminotransferase, alkaline phosphatase, blood urea nitrogen, creatinine, phosphate,
calcium, total protein, albumin, globulin, total bilirubin and amylase as per manufacturer’s instructions.
2.10 Determination of bone linear growth and estimation of bone density
The femur and tibia from the right leg of each rat were cleaned of non-calcified tissue and oven-dried (Salvis®, Switzerland)for five days at a temperature of 50˚C. Bone dry mass was measured using a balance (Presica 310M Laser, Johannesburg, South Africa) whilst bone length was measured using venier calipers (Hi-impact, Dejuca, South Africa) were used to measure bone length. Tibia length was measured between the tibia head and medial malleolus.Femur length was measured between the greater trochanter and medial condyle. The Seedor index was used to estimate bone density and was calculated using the
following formula;
Seedor index = mass of bone (mg)/length of bone (mm) (Seedor, 1991, Almeida et al., 2008).
Radiographs of the bones were also taken to subjectively assess bone density using a Fuji film X-ray machine (Industrial X-ray film FR; Fuji Photo Film Co., Ltd, Tokyo, Japan). The bones were placed on a photographic plate, 1 metre away from the X-ray light source set at 4.8 kVp, 0.71 mA per plate for 10 seconds.
26 2.11 Statistical analysis
All data are expressed as mean and standard deviation and were analysed using Graph Pad Prism 5 (Graph Pad Software, San Diego, California, USA). Statistical significance was set at 5%. Linear growth, visceral organ mass, concentrations of circulating metabolites, liver lipids and general health profile markers were analysed using one-way analysis of variance (ANOVA). Body mass changes, oral glucose tolerance tests and food and fluid intake were analysed using the two-way repeated measures ANOVA with treatment and time as main effects. Unpaired student t-tests were used to analyse sex differences of the above
parameters. The Bonferroni post hoc test was used to detect differences between and or within groups whenever the ANOVA showed significant differences or significant main effects.
27
CHAPTER 3 : RESULTS
28
3.1 Effect of neonatal intake of ursolic acid on growth performance 3.1.1 Body mass measurements
Figure 3.1 shows the induction, weaning and terminal masses of male (A) and female (B) rats across all the treatment groups. The treatments given in the neonatal phase had no adverse effects on growth of the rats across the treatment groups (P˃ 0.05). There was significant growth, from induction to weaning and from weaning to termination within all treatment groups in both sexes (P˂ 0.0001). In males, a late fructose hit, that is, rats receiving dimethylsulphoxide (DMSO) in the neonatal phase and fructose in adulthood (DMSO+FW) had increased terminal mass compared to rats receiving DMSO neonatally and plain water in adulthood (DMSO+ PW) (main effects of time (P˂ 0.0001), treatment (P= 0.079), and their interaction (P= 0.0073)). No differences were observed in rats receiving an early fructose hit (fructose neonatally and water in adulthood; FR+FW) as well as a double fructose hit (fructose neonatally and in adulthood) (P> 0.05). Ursolic acid (alone and in combination with fructose) had no effects on body mass (P˃ 0.05).
In females, UA administration alone promoted increases in terminal body mass; rats receiving UA neonatally with fructose in adulthood (UA+FW) had significantly higher terminal masses than the group receiving dimethylsulphoxide in the neonatal phase and fructose in the third phase (DMSO+FW) (main effects of time (P˂ 0.0001) treatment (P= 0.21), and their interaction (P= 0.37)). No differences were observed in rats receiving a late fructose hit as well as a double fructose hit (fructose neonatally and in adulthood; FR+FW) (P> 0.05). A comparison of the sexes revealed that male rats had significantly greater body mass gains than female rats across all treatments (P˂ 0.0001).
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Induction Weaning Termination
0 200 400 600 800 DMSO + PW DMSO + FW UA + PW UA + FW FR + PW FR + FW UAFR + PW UAFR + FW *** *** B o d y M a s s ( g )
Induction Weaning Termination
0 200 400 600 800 *** *** B o d y M a s s ( g ) A B
Figure 3.1: Induction, weaning and terminal masses of male (A) and female (B) rats given different treatments.
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All data presented as mean ± standard deviation. *** = significant growth from induction to weaning and from weaning to termination (P< 0.0001). μ = significantly greater terminal masses in male rats receiving DMSO+FW than those receiving DMSO+PW (P˂ 0.05). γ = significantly greater terminal masses in female rats receiving UA+FW than those receiving DMSO+FW (P˂ 0.05). DMSO + PW =10 mg/kg b.w dimethylsulphoxide in neonatal phase + plain water in adulthood (n=14; 8 M, 6 F); DMSO + FW =10 mg/kg b.w dimethylsulphoxide + 20% fructose in drinking water (n=13; 7 M, 6 F); UA + PW =10 mg/kg b.w ursolic acid + plain water (n=14; 7 M, 7 F); UA + FW =10 mg/kg b.w ursolic acid + 20% fructose in drinking water (n=13; 7 M, 6 F); FR + PW =10 mg/kg b.w fructose + plain water (n=13; 6 M, 7 F); FR + FW =10 mg/kg b.w fructose + 20% fructose in drinking water (n=14; 6 M, 8 F); UAFR + PW =10 mg/kg b.w ursolic acid and fructose + plain water (n=14; 7 M, 7 F); UAFR + FW =10 mg/kg b.w ursolic acid and fructose + 20% fructose in drinking water (n=12; 6 M, 6 F).